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Electroforesis Cuantificación Band - Un Arte o Una Ciencia
Electroforesis Cuantificación Band - Un Arte o Una Ciencia
Electroforesis Cuantificación Band - Un Arte o Una Ciencia
There are so many things to consider when trying to accurately quantify and compare
bands in an electrophoresis gel that it has often been described as more of an art than a
science. The purpose of this article is to identify and discuss the relevant points so that
scientists may be sure that the method of quantifying band material they are using or
intend to use is as accurate as is feasible. Electrophoresis is a powerful, useful and widely
used technique, but is nevertheless fraught with difficulties. The following points include
some of the things that need to be considered for the accurate quantifying of bands. The
discussions on each point are intentionally general and specific reference to any particular
electrophoresis techniques or products has been avoided. This has the effect making the
process appear over complicated. However, once the elements that relate to particular
applications are identified electrophoresis band quantification can be shown to be a
relatively simple science.
generally limiting in the range of analyses that could be performed. For these reasons it is
currently generally preferred to use an instrument that is capable of acquiring a gel image
that can then be analyzed using image analysis techniques.
The type of image capture device that is used will depend on the kind of visualization
method that is employed. For example DNA / ethidium bromide gels visualized with UV
light will often be captured for analysis using a CCD Video camera system. Images of
radiolabelled gels or blots may be obtained using a Phosphor imaging device or a direct
radiation imager. Similarly, fluorescence or chemi-luminescence visualization methods
may make use of photosensitive instruments designed to produce images from such gels
or blots. In recent years the use of commercial scanners has become a popular method of
acquiring images from stained gels, blots or autoradiograms.
Whichever system is used it is important that the calibration method, if any, is known and
understood. Despite the claims of manufacturers there are very few image capture
systems that are innately linear in response. Those devices that are demonstrated to
produce a linear response are often calibrated to provide that response. This is fine if you
are using the machine in precisely the same way as the manufacturer intended. Any
deviation may require an alternative calibration method unless the demands on the
analysis are not strict and therefore do not require calibration. Also it is often not
necessary to calibrate for non-linearity if alternative quantity calibration methods are used
whereby a series of spots or bands of known value are used to produce a calibration
curve.
Traditionally many machines calibrate transmission readings to Optical Density. As light
passes through an object the degree of absorption is logarithmically related to the density
of the object. Generally the following calculation is used to provide a linear response: Optical Density = -log10[(255-raw pixel value)/255]
New ISO Transmission Density Standards used during the calibration of instruments
designed to examine the density of photographic products with transmitted light make use
of diffuse density which, in effect recognizes the use of diffusers in densitometers. Optical
Density values differ from diffuse density values by a simple multiple of around 1.414.
However, this is only important for those few applications that require absolute values.
Most electrophoresis densitometry applications are examining relative values rather than
absolute values, in which case the difference is academic.
x Lane Percentage: The value of each band is expressed as a percentage of the sum of
all the material in the lane including inter band material.
x Lane normalization: Each lane is given an overall value and each band is expressed as
a proportion of that value.
x Normalization to an individual band: A matched band across all the lanes (one that is
deemed to be theoretically unchanging) is given a value and the values of all the other
bands in each lane are adjusted in accordance to the matched band having the given
value.
7 Quality Reporting
Naturally all data must be reported in some way and it should be a simple matter to
present the image, data, associated graphics and calibration methods within a report. For
some applications automated reporting within the analytical software is desirable,
especially when considering the requirements of Good Laboratory Practice. In addition it
should be possible to use copy and paste methods so that scientists can structure their
own reports in the way they find most suitable for their applications.
9 Resolution
Sometimes, with certain types of gels containing many merged bands over an uncertain
level of background, it will be found that a scientific determination of error limits produces
margins that are unacceptably broad. In such cases it is tempting to desire equipment with
higher resolution. Generally this is not the answer because improved resolution will often
only help a little, not enough to overcome the unacceptably broad margins of error. For
those applications where a high resolution is necessary and desirable, the scientist should
be very careful of misquoted figures. Many scanners have very tempting quoted levels of
resolution in dots per inch (dpi) far in excess of scientists needs. However these figures
do not account for any averaging between pixels. As a general rule of thumb for
electrophoresis applications scanning above 400 dpi does not tend to improve the
resolution sufficiently to reduce margins of error significantly. Imaging laser densitometers
have a similar resolution (80-90Pm). The resolution of CCD camera systems is a function
of the number of light sensitive points on the CCD chip and the size of the area being
photographed. At best this results in a similar resolution to standard scanners. Therefore
the only way to significantly improve resolution is to purchase a microdensitometer, a drum
scanner, or a high resolution CCD camera system. Whether such instruments would help
is dependent on the application.
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